Detection of SARS-CoV-2 N protein using AgNPs-modified aligned silicon nanowires BioSERS chip

The SARS-CoV-2 (COVID-19) pandemic had a strong impact on societies and economies worldwide and tests for high-performance detection of SARS-CoV-2 biomarkers are still needed for potential future outbreaks of the disease. In this paper, we present the different steps for the design of an aptamer-based surface-enhanced Raman scattering (BioSERS) sensing chip capable of detecting the coronavirus nucleocapsid protein (N protein) in spiked phosphate-buffered solutions and real samples of human blood serum. Optimization of the preparation steps in terms of the aptamer concentration used for the functionalization of the silver nanoparticles, time for affixing the aptamer, incubation time with target protein, and insulation of the silver active surface with cysteamine, led to a sensitive BioSERS chip, which was able to detect the N protein in the range from 1 to 75 ng mL−1 in spiked phosphate-buffered solutions with a detection limit of 1 ng mL−1 within 30 min. Furthermore, the BioSERS chip was used to detect the target protein in scarcely spiked human serum. This study demonstrates the possibility of a clinical application that can improve the detection limit and accuracy of the currently commercialized SARS-CoV-2 immunodiagnostic kit. Additionally, the system is modular and can be applied to detect other proteins by only changing the aptamer.


Introduction
In late 2019, a novel severe acute respiratory syndrome (SARS-CoV-2) virus of unknown origin appeared in Wuhan (China). 1 SARS-CoV-2 affects the lungs damaging them severely and oen causing lung failure, 2 leading to death.To date, the worldwide toll of death is around 6.98 million, but it could be much higher since all the deaths were not reported.This new coronavirus caused the disease of COVID-19 around the world leading to a global pandemic. 3The SARS-CoV-2 infection rate reached alarming levels due to its easy transmission through direct contact via respiratory droplets from an infected person or by touching surfaces contaminated with the virus. 4,5Following its discovery, countries worldwide implemented strict safety measures, such as border closures, mandatory self-quarantine, and the shutdown of public and crowded spaces.These measures have signicantly impacted global economic growth, leading to the bankruptcy of numerous businesses.This crisis has had a devastating effect on the global economy, resulting in losses exceeding $16 trillion. 6This high rate of transmission has prompted scientists and researchers worldwide to focus on developing new detection methods alternative to cumbersome real-time polymerase chain reaction or modifying existing ones to effectively identify the virus in its early stages of development.
Detecting the virus at early stages can help mitigate its widespread impact.
Besides 16 non-structural proteins (NSPs1-16), 7 the main four proteins of the novel coronavirus are the spike (S), the membrane protein (M), the envelope protein (E), and the nucleocapsid protein (N), which is the only core protein.The latter is the most immunogenic one triggering a high immune response among infected individuals. 8The N protein is well studied in SARS-CoV-1 (ref.9) and it is associated with the viral RNA function and plays a main role in viral transcription. 10hile the S protein is currently the primary target for COVID-19 biosensing applications due to its indispensable functions, recent studies have shown that in some cases the N protein is more sensitive than the S protein for detecting early infection. 11oreover, the latter is subject to several mutations in the RBD zone such as K417N, S477N, E484A, Q493K, G496S, Q498R, etc., which will require preparing a specic detector for each mutant.Thus, targeting the N protein seems a more forward approach for COVID-19 detection in sera and nasal swabs using rapid antigenic tests.
Aptamer-based biosensors employ aptamers as recognition elements linked to a physical signal transducer, translating the binding interaction between the aptamer and its target into a detectable signal, thereby facilitating precise and selective identication of particular analytes. 12,13The use of aptamers, which are single-stranded DNA or RNA oligonucleotides, in biosensing as specic receptors for quick screening of disease biomarkers has garnered signicant interest.To replace antibodies, which are oen costly and subject to degradation, aptamer-based biosensing devices are used. 14,157][18][19] Their primary advantages are high selectivity, affinity, and stability, which enable them to distinguish between protein isoforms and splice variants and increase the potential for numerous applications, even in multiplex discovery platforms. 20Additionally, the longevity of a commercial aptamer-based detection kit is increased by the stability of aptamers, which can even be regenerated aer denaturation.
Among several sophisticated analytical instruments, surfaceenhanced Raman spectroscopy is a powerful analytical tool that can be used in the detection of different biological molecules. 21ERS can provide a precise ngerprint spectrum even in complex samples with very low concentrations.Also, it is a nondestructive method that needs a short analysis time and provides a high sensitivity and low detection limit.In recent work, we have successfully designed a SERS biosensor to detect prostate-specic antigens using an aptamer tethered to silicon nanowires (SiNWs) decorated with silver nanoparticles (AgNPs). 22Lysozyme is a food allergen that could contaminate food during the processing step.Boushell et al. designed a SERS-based sensor for the detection of lysozyme on foodhandling surfaces. 23Negri et al. reported the design of two different SERS platforms for the detection of Inuenza A through the detection of its nucleoprotein using an aptamer. 24,25hen et al. reported the SERS detection of the whole Inuenza A/H1N1 virus using an aptamer targeting the viral hemagglutinin surface protein and popcorn-like gold nanostructures for signal enhancement. 26n this work, we developed a SERS-based aptasensor platform capable of detecting the SARS-CoV-2 N protein with a high sensitivity.A specic DNA aptamer for the N protein is used as a receptor and an AgNPs/SiNWs substrate for signal transducing.The SiNWs fabrication and their decoration with AgNPs are achieved by a relatively simple wet-lab chemical processing method. 22A quantitative analysis of the SARS-CoV-2 lysate is performed by monitoring the change in the SERS peaks intensity and area caused by the new binding between the aptamer DNA attached to the AgNPs/SiNWs surface and the N protein in the SARS-CoV-2 virion.This biosensor enables detecting the target with a limit of detection of 1 ng mL −1 in less than 30 min.This methodology is costly effective, robust, rapid, and requires very small sample volumes, which can help in the diagnosis domain of infections at very early stages.
All the solutions were prepared using deionized water produced by a Millipore system.The 5 0 -thiolated anti-N specic DNA aptamer (5 0 -aaa aac gcg cgt att cct tag ggg cac cgc tac acg cgc g-3 0 ) was acquired from Biomers (Germany).The sequence was puried by HPLC to ensure the maximum purity and the reproducibility of the test.This DNA aptamer was recently discovered by Zhang et al. for the targeting of recombinant COVID-19 nucleocapsid protein of SARS2-CoV-2. 27Membrane protein (M protein) and recombinant SARS-CoV-2 spike glycoprotein (S protein) were purchased from Abcam.The human blood serum used in this work was purchased from Sigma-Aldrich (product ref.H4522).

Production of a recombinant N protein
A His-tagged recombinant form of the COVID-19 N was expressed in E. coli.Briey, the cDNA encoding for the COVID-19 N protein was rst generated by reverse transcription using the Superscript II RT kit (Invitrogen).COVID-19 N encoding gene was later amplied using two primers, NF1 5 0 -cagggattccgatgtctgataatggaccccaaa-3 0 and NR1 5 0 -ctcgtcgacggcttgagttgagtcagcactgc-3 0 .The PCR product was later digested with BamH1 and Sal1 restriction enzymes and ligated to the pET26 vector, linearized by BamH1 and Xho1 enzymes.A recombinant construct was later used to transform the BL21 E. coli strain for protein expression and purication in native conditions.The recombinant protein was rst puried on a Nickel-Sepharose column and then on a G75 Sepharose gel ltration column.Protein purity reached more than 95%, as assessed on SDS-PAGE gel.

Instrumentation
Raman measurements were performed on a Micro Raman HORIBA system (LabRAM HR800) at room temperature (RT) with a helium-neon laser (l = 632.8nm) and a power of 2.4 mW at the sample surface, with a 100× microscope objective.
The morphological properties of the surface and the crosssection of the samples were analyzed using a JEOL JSM 7100F thermal eld emission electron gun scanning electron microscope (SEM).Composition analysis of the cross-section of the samples was performed using a conventional HITACHI FLEX SEM II microscope equipped with an energy-dispersive detector (EDX).
The absorbance spectra were obtained at RT in the wavelength range of 250 to 400 nm using an UV-Visible-NIR spectrophotometer (PerkinElmer Lambda 950).

Elaboration of the BioSERS platform
The design of the BioSERS platform for detecting the N protein involved a series of sequential steps.Initially, SiNWs were fabricated in 2 steps using a metal-assisted chemical etching technique.This involved treating lightly N-doped Si (100) wafers with an HF solution. 28To deposit AgNPs onto the SiNWs, we employed the chemical reduction of Ag + using Si-H groups.The optimization of these parameters had already been carried out in a previous work. 22To prevent the non-specic binding of analytes to the silver surface, a critical step in our protocol involved the strategic use of a selfassembled monolayer (SAM).In our previous work, 22 we employed MCH as a SAM, and this was applied before introducing the aptamer onto the SERS substrate.However, in the current study, we introduced a key modication.Instead of applying the SAM before the aptamer, we opted for a different SAM composed of cysteamine, and this step was performed aer the attachment of the aptamer.This decision was driven by a two-fold consideration.Firstly, cysteamine, with its unique molecular structure, served as an effective blocking agent, preventing non-specic adsorption of analytes onto the bare silver surface. 29Secondly, placing the cysteamine SAM aer the aptamer attachment underscored the adaptability and versatility of our SERS substrate.By immersing the substrate in a solution of cysteamine hydrochloride aer the aptamer was in place, we ensured that the SAM formation precisely complemented the functionalization with the aptamer.This sequence of steps was carefully designed to optimize the performance of our aptasensor.To achieve this, the thiolated DNA aptamer was tethered to the surface of the AgNPs by incubating the sample into a 0.5 mL solution containing 1 mM of anti-N aptamer dissolved in PBS for 4, 8, 12, and 16 hours at RT. Next, we immersed the SERS substrate in a solution of 10 −3 M cysteamine hydrochloride dissolved in 10 mM PBS solution. 30Subsequently, the sample was removed from the solution, washed twice with PBS and deionized water, and dried under a nitrogen ow.In the nal stage, the aptasensor (cysteamine/anti-N/AgNPs/SiNWs) underwent incubation in different concentrations of N protein dissolved in a PBS solution at a pH of 7.4.The incubation time for this step ranged from 20 to 30 minutes. 31Finally, the prepared substrates were thoroughly washed with deionized water and dried before Raman measurement.Fig. 1 schematically depicts the preparation of the BioSERS chip.

Surface characterizations
In the context of SERS, the uniform distribution of metallic nanoparticles on the substrate plays a pivotal role in determining the reliability and reproducibility of the measurements.Fig. 2A-C provide insights into the surface morphology of the AgNPs/SiNWs substrate, showcasing both the crosssectional and top-view perspectives.The uniformity of the material is of paramount importance for SERS efficacy.In SERS, the enhancement of Raman signals is highly dependent on the localized surface plasmon resonance (LSPR) generated by metallic nanoparticles.The spatial arrangement and density of these nanoparticles inuence the "hot spots" on the To ascertain the functionalization with silver nanostructures, elemental composition by EDX (Fig. 2D and E) and UV-visible absorbance (Fig. 2F) spectroscopies were carried out before and aer treatment of the silver nanoparticles.The UVvisible absorbance spectrum of the metalized substrate shows a peak around 320 cm −1 attributed to the surface plasmons due to the presence of the silver nanoparticles.The EDX elemental analysis evidenced the presence of AgNPs and showed the penetration of the metallic nanoparticles into the pores of the SiNWs.One can see from the results, the presence of % Si = 66.09, % O = 12.35, and % Ag = 21.65.The presence of new peaks at energy above 3 keV related to La emission of silver, conrms the success of the introduction of the AgNPs.The EDX spectrum performed on the cross-section indicated that the silver mass percentage from the surface to a depth of 2.37 mm was approximately 21%.Then, the percentage gradually decreases as the depth increases becoming zero for depths beyond 4.6 mm.The results provide evidence that the silver nanoparticles successfully modied the silicon nanowires.
Raman spectroscopy was employed to further conrm these modications.Specically, the overlap of the spectra obtained for the silicon nanowires at each modication step indicates the existence of organic groups derived from the aptamer and the cysteamine-blocking group (Fig. 3).

Optimization of the parameters
3.2.1.Aptamer concentration.Several key parameters are expected to affect the response of the BioSERS chip such as the incubation time of aptamer with the AgNPs/SiNWs substrate or the time necessary for the binding of target to the aptamer receptor.Since the functionalization of the surface of silicon nanowires with the aptamer is important for the detection step and to achieve high sensitivity, we choose to incubate the AgNPs/SiNWs substrate in a rather large amount (0.5 mL of 0.5 mM, 1.0 mM or 5.0 mM) of the aptamer solution for 4 hours of incubation using the Raman signature of the N protein as a probe to assess the sensitivity to it.Fig. 3A depicted the different Raman spectra, revealing the emergence of novel modes subsequent to incubating the AgNPs/SiNWs substrate in different concentrations of the aptamer solution.The presence of new SERS modes at 500-800 cm −1 , 800-1200 cm −1 , and 1200-1600 cm −1 alongside the second-order silicon mode at 960 cm −1 indicates the successful tethering of the aptamer on the surface of silver nanoparticles.Upon careful comparison of the intensity of the modes for the three concentrations, it is evident that the medium concentration yielded the most distinct spectrum.Furthermore, the Raman spectra obtained at different aptamer concentrations revealed an interesting non-linear response.Specically, lower intensity peaks were observed for the 0.5 mM concentration, which increased at 1 mM, and then decreased for 5 mM.This non-linear response may be inuenced by factors such as the formation of a thicker layer of analyte at higher concentrations, impacting molecular orientation and interaction with the metallic surface.Similar observations have been reported in previous studies, where the presence of a thick layer of analyte molecules hindered the SERS effect, leading to decreased signal intensities.For instance, in a study by Yang et al., 32 it was found that at micromolar concentrations, the SERS effect was hindered by a thick layer of analyte molecules formed on the surface, potentially resulting in bulk Raman scattering rather than SERS.To address this issue, the gold surface was backlled with a self-assembled monolayer of hexanethiol immediately aer aptamer immobilization.This SAM prevents nonspecic adsorption of analyte molecules by blocking access to the surface and improves the efficiency of aptamer capturing.We therefore used the 1 mM concentration for the rest of our study.
3.2.2.Filling agent addition.We further added cysteamine to block the surface and prevent the non-specic adsorption of the target on the bare surface of AgNPs.The modied surface by cysteamine (10 −3 M for 30 minutes) was evaluated by Raman measurements.Spectrum (d) in Fig. 3B showed the appearance of a new mode related to cysteamine with very high intensity at 640 cm −1 which is attributed to the C-S bond. 33Furthermore, the main modes of the aptamer decreased in their scattered intensities such as the mode at 720 cm −1 .The slight intensity decrease of aptamer vibration modes can be explained by the exchanging of the aptamer with the cysteamine and also the concentration difference between the two of them (10 −3 M for cysteamine vs. 1 mM for aptamer).
Due to its composition as a single-stranded DNA, the aptamer displays distinct and prominent Raman signatures that are typical of oligonucleotides.5][36] In general, the Raman spectrum of DNA is divided into three regions that correspond to specic vibrational modes, including the vibrations of the DNA bases (500-800 cm −1 ), phosphate and sugar (800-1200 cm −1 ), and nally those of the DNA skeleton (1200-1600 cm −1 ), which are particularly inuenced by the secondary structure of DNA. 35In particular, the main vibrational modes detected for the aptamer are outlined in Table 1.
For longer incubation durations, the SERS spectra showed that the intensity of peaks increased but we also lost resolution since the signal width increased as well, probably due to a steric hindrance and the presence of a large amount of DNA strands on the surface of silver nanoparticles.The observed disappearance of the signal aer 16 hours of incubation is attributed to the high and excessive adsorption of the aptamer on the surface of the nanoparticles.Prolonged incubation times can lead to saturation and overcrowding of the surface. 37This phenomenon, while inuencing the SERS signal strength, also highlights the importance of optimizing incubation times for achieving a balance between effective functionalization and maintaining signal stability.Hence, incubation for 8 hours showed the best result and was selected further to carry out the rest of the work.
DNA mode observed at 720 cm −1 , attributed to adenine ring stretching (Table 1), exhibited a signicantly high intensity and distinguished itself from the others.Additionally, it is not included in the amide II and amide III regions so it can be used to assess the binding of the target protein, which will appear in the region of 1100 cm −1 to 1650 cm −1 .
3.2.4.Protein recognition.Since the amount of the target protein tethered to the surface of the BioSERS chip is dependent on the aptamer quantity present on the surface of AgNPs, we examined the effect of the aptamer incubation time (2, 4, 8, and 16 h) on the N protein.Also, it was intended to verify whether the duration of 8 h, arbitrarily posed in the preliminary experiments, was the adequate one.Fig. 3D showed the SERS spectra of N protein (15 ng mL −1 ) incubated with the bioSERS chip for different durations.For short incubation times (2 and 4 h), the spectra do not clearly show the stretching modes of the target analyte.For longer incubation times, Raman vibration modes of amides are visible, indicating the presence of the N protein.
As we can see, the peaks related to cysteamine (615-680) cm −1 were still visible, which conrmed that the surface is still blocked, the observed peaks are due to the target protein bound to the aptamer.Furthermore, new intense peaks appear at 1020 and 1400 to 1480 cm −1 and in the ranges of 1131 to 1190 cm −1 , 1200 to 1380 cm −1 and 1500 to 1620 cm −1 .9][40] The peaks in the 1131-1190 cm −1 range are related to the nitrogenous bases of the DNA nucleotides.Indeed, the 1087 cm −1 mode is related to the n CO stretching in amides and proteins, the 1136 cm −1 mode is assigned to the n NH 2 of the guanine, the 1180 cm −1 mode is attributed to deoxythymidine, the 1173 and 1176 cm −1 modes represent the n CH of the tyrosine. 35,38,390][41] The intensities of these modes increased monotonously upon time increased from 2 h to 8 h and then decreased for 16 h.The optimal incubation time of AgNPs/SiNWs in aptamer was set to 8 h to carry out the experiment since it has shown the best results both for aptamer/AgNPs/SiNWs and N-protein/cysteamine/aptamer/AgNPs/SiNWs.

Performance of the BioSERS chip
Fig. 4A displayed the SERS spectra, before and aer adding the target protein to the cysteamine/aptamer (8 h)/AgNPs/SiNWs bioSERS chip.The N protein concentrations ranged from 1 to 75 ng L −1 .The protein addition induced the appearance of new peaks, which increased with the target increasing concentrations.The latter indicated that the amount of N protein bound to the aptamer is becoming important.The main vibration modes are the amide III in the range 1200-1380 cm −1 and the amide II in the range 1500-1620 cm −1 .The peak area of the amide II vibration located at the range of 1500-1620 cm −1 was used to plot the calibration as a function of the concentration (Fig. 4B).The calibration plot is linear in the range from 1 to 75 ng mL −1 , following eqn (1): Amide II area (a.u.) = 641.72[Nprotein] (ng mL −1 ) + 24.78 (1) The correlation coefficient of the linear curve is R 2 = 0.989 and the limit of detection is estimated to be the lowest detection concentration 1 ng mL −1 , calculated from the 3 × S b /m criterion, where m is the slope of the calibration curve and S b was estimated as the standard deviation of three different measurements recorded for the lowest analyte concentration.
3.4.Selectivity and shelf-life 3.4.1.Selectivity and specicity.Selectivity and specicity play a crucial role in determining the performance and reliability of any detection method.To evaluate these important characteristics, we considered the S protein and M protein of the coronavirus as competing proteins for the target protein.
The selectivity and specicity tests were carried out under identical conditions to that used the detect the target protein, where each protein (S, M, and N) was added at a concentration of 33 ng mL −1 .
In Fig. 4C, the peak area of amide II for each type of protein was plotted.It can be observed that a distinctive difference in the intensity of peaks within the BioSERS chip was exposed to 33 ng mL −1 of different examined proteins.Specically, the N protein led to a noticeable increase in SERS peak intensity for the modes associated with proteins, whereas the S and M proteins did not cause any signicant changes.This outcome demonstrates the exceptional selectivity of the biosensor towards the N protein.Furthermore, to assess the specicity of the system, the response of the chip in the presence of the two aforementioned proteins in the presence and absence of the N Fig. 4 (A) SERS spectra of bioSERS chip aptamer/cysteamine/AgNPs/SiNWs in the presence of different protein concentrations 1 ng mL −1 (c), 5 ng mL −1 (d), 10 ng mL −1 (e), 15 ng mL −1 (f), 20 ng mL −1 (j), 30 ng mL −1 (h), 50 ng mL −1 (i) and 75 ng mL −1 (j), (B) calibration plot displaying the amide II mode (1500 to 1630 cm −1 ) area versus the concentration of the target protein.(C) Analytical response of the aptasensor toward different proteins: N (33 ng mL −1 ), S (33 ng mL −1 ) and M (33 ng mL −1 ); amide II mode area for each type of analyte.(D) Storage stability of the aptasensor stored for 57 days at 4 °C and its response in presence of 33 ng mL −1 of N protein recorded periodically.
protein was examined.Interestingly, no signicant response is obtained in the absence of the N protein.This nding provided further evidence of the chip's ability to specically detect the N protein while maintaining minimal interference from other proteins.
3.4.2.Shelf-life.To assess the storage stability of the bio-SERS chip, it was carefully monitored approximately for two months when it was stored in a refrigerator at 4 °C.During this time, response was periodically recorded both with and without the presence of the N protein.As depicted in Fig. 4D, the chip showcased a consistent and stable signal even aer 57 days of storage (longer durations were not assayed), whether in the absence or presence of 33 ng mL −1 of N protein.

Applicability in complex matrix
To better assess the selectivity of the chip, its performance was tested on complex samples, specically using human blood serum from a healthy donor.The serum contains an assortment of compounds including proteins and peptides like albumins, globulins, lipoproteins, enzymes, and hormones.Additionally, vital nutrients such as carbohydrates, lipids, and amino acids are present, as well as electrolytes, organic waste, and a variety of small organic molecules, both suspended and dissolved.In this study, 150 ng mL −1 of N protein was introduced into the 10×diluted serum with phosphate buffer saline solution to decrease its viscosity.Using the same detection procedure employed with the buffered solutions, Fig. 5 illustrated the response in the presence of the diluted serum, as well as aer the addition of N protein to the mixture.
Particularly amide II group is visible for the spiked serum sample while it is completely absent for the diluted serum of the healthy subject.This nding highlights the ability of the Bio-SERS chip to distinguish and detect the N protein even in complex biological samples such as blood serum.

Comparison with other works
The BioSERS chip developed for the N protein detection exhibited superior analytical performance compared to previously reported works, as indicated in Table 2.Although its detection limits and dynamic ranges are slightly lower than those of the electrochemical and colorimetric biosensors reported in ref. 42 and 43, and its detection time is shorter than that of SERS biosensors mentioned in ref. 44-46, this device offers the advantage of being easier to manufacture and having a longer shelf life of up to 2 months.Additionally, it outperforms in terms of detection limit the biosensors utilizing

Discussion
The paper reports the design and characterization of a BioSERS chip for the detection of the N protein, a key component of the coronavirus-2, which originated the COVID-19 pandemic in 2019.The study involves several crucial steps, including surface characterizations, optimization of operational parameters, performance evaluation, selectivity and specicity testing, shelflife assessment, and applicability in spiked buffered solutions, complex matrices, and real samples.The surface characterizations demonstrated the successful modication of silicon nanowires with silver nanoparticles and the anti-nucleocapsid aptamer.The consistent and uniform structure of SiNWs, the spherical shape of AgNPs, and the conrmation of the successful introduction of AgNPs through SEM images, EDX analysis, and UV-visible spectroscopy provided a strong foundation for the subsequent experiments.Raman spectroscopy further conrmed the modications, indicating the presence of the signature of the organic groups derived from the aptamer building blocks and cysteamineblocking agent.
We further examined various parameters to optimize the sensitivity of the BioSERS chip.Indeed, the aptamer concentration testing revealed that a medium concentration (1 mM) yielded the most distinct spectrum, indicating successful aptamer tethering.The addition of cysteamine was benecial since it efficiently blocked the surface to prevent the non-specic adsorption of the target biomolecule, and an 8 hours incubation period for the aptamer was determined as the optimal duration.This duration provided a balance between signal intensity and resolution, making it suitable for further experiments.
The BioSERS chip demonstrated excellent performance in detecting the nucleoprotein.The calibration plot shows a linear relationship between the amide II peak area and N protein concentration varying from 1 to 75 ng mL −1 , with a low detection limit of 1 ng mL −1 .Selectivity and specicity studies revealed that the device was able to selectively detect the N protein, distinguishing it from competing S and M proteins from the same virus and maintaining minimal interference.The chip also exhibited remarkable stability over a 57 days storage period.Longer storage durations were not assayed.
The applicability of the BioSERS chip in complex matrices was validated using spiked diluted serum, where it successfully detects the N protein even in the presence of various serum components.These results highlighted the device's potential for real-world applications, especially in clinical diagnostics and disease monitoring.
Comparative literature analysis showed that the developed BioSERS chip outperformed several previously reported sensing methods in terms of detection limit, ease of manufacturing, and shelf life.While some other methods exhibited slightly lower detection limits and wider dynamic ranges, the advantages of the BioSERS chip in terms of simplicity and longevity made it a promising candidate for practical use.
In addition to the excellent performance demonstrated by the BioSERS chip in detecting the nucleoprotein of SARS-CoV-2, further enhancements in sensitivity and detection capabilities can be achieved through the integration of surface-enhanced Raman spectroscopy with rolling circle amplication. 50The combination of these two techniques holds promise for amplifying the signal generated by the target biomolecule, thereby improving the chip's overall sensitivity, and lowering its limit of detection.Future research efforts could focus on integrating RCA amplication into the BioSERS chip platform to further enhance its sensitivity and broaden its applicability in detecting viral proteins like the N protein of COVID-19, even in complex biological matrices.This integrated approach would contribute to the ongoing efforts to develop highly sensitive and rapid diagnostic tools for combating infectious diseases.

Conclusion
The paper presents a comprehensive study on the development and characterization of a BioSERS chip for the sensitive and selective detection of the N protein.Its superior analytical performance and practical advantages position it as a valuable tool in the eld of disease diagnostics and monitoring, particularly in the context of COVID-19.This technique enables detecting SARS-CoV-2 N protein with a detection limit of 1 ng mL −1 within 20 to 30 min.The ndings pave the way for further research and potential commercialization of the developed BioSERS chip for widespread use in healthcare and related applications.

Fig. 1
Fig. 1 Schematic illustration of the step-by-step preparation of the BioSERS sensing chip and detection of the N protein.

Fig. 5
Fig. 5 BioSERS response to the healthy blood serum before and after the introduction of N protein into the mixture.

Table 1
Assignment of the SERS peaks of the aptamer and SARS COV2 N protein

Table 2
Comparison of different detection strategies for SARS-CoV-2 virus a assay and SERS spectroscopy, as well as some of the electrochemical biosensors mentioned in ref.44-48.